Spatiotemporal Expression of Burkholderia pseudomallei Genes

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Burkholderia pseudomallei, the causative agent of melioidosisis, is classified as a Category B potential bioterrorism agent because of its historical use in several wars. It affects American servicemen and travelers, and exists in some parts of the United States, including Hawaii. Knowledge about the spatial and temporal gene expression during cellular infection is crucial to the understanding of B. pseudomallei molecular pathogenesis.

Central hypothesis

B. pseudomallei, as it encounters uniquely different intracellular environments, undergoes differential gene expression at each stage of cellular infection.

Objective

To thoroughly identify in vivo expressed and essential genes and to characterize the expression of these genes with respect to temporal and spatial resolution during infection of eukaryotic cells.

Specific Aim 1

Identify and assign in vivo expressed B. pseudomallei genes on a temporal scale.

Hypothesis: Once whole macrophage culture infection initiates, differential gene expression identified at various times will reflect genes required for different stages of cellular infection.

Rationale: Identifying genes differentially expressed on a temporal scale during host-cell infection will yield insight into the pathogenic mechanisms of B. pseudomallei.

Experimental Plan: We will utilize the available B. pseudomallei whole genome array from the NIAID Pathogen Functional Resource Center at the J. Craig Venter Institute. We will infect the mouse macrophage cell line RAW264.7 with B. pseudomallei strain K96243, kill extracellular bacteria, and bacterial RNA will be isolated after lysis of macrophage culture at various times for microarray and real-time RT-PCR studies.

Specific Aim 2

Identify essential B. pseudomallei genes for macrophage infection and assign the expression of these genes on a spatiotemporal scale.

Hypothesis: Only a subset of in vivo expressed genes in aim 1 are essential for the progression of cellular infections.

Rationale: Since the majority of gene expressed in aim 1 may not be essential for intracellular infection, the negative selection strategy in aim 2 will identify genes directly essential for the intracellular infection process.

Experimental Plan: A high-throughput signature-tagged mutagenesis approach will be utilized with four fluorescent protein tags (red, yellow, green, and cyan) each individually located on a transposon. Each well of a 96-well plate will contain four different colored bacteria (i.e., 4 different transposon mutants), which will be infected into a single well of a 96-well plate of cultured macrophage. Hence, each 96-well plate of cell culture will allow screening of 384 transposon mutants. Screening will be performed to detect the lack of any four types of fluorescent plaques in each well. We will confirm expression of these genes by live cell imaging and real-time RT-PCR.